Method of rapidly detecting microorganisms



June 24, 1969 R. e. SANDERS 3,451,893 METHOD OF RAPIDLY DETECTINGMICROORGANISMS Filed Sept. 27, 1966 Sheet of 4 FIG )5. 3 f5 a 2 Z O. "2I: f. o I S U '5 O 3 U E 8 ENZYME CONCENTRATION (Grams Per MiIliIiIer)Fla 2 C I g I x Lu '6 I I Q l- 40 I 2 I E I I I I "6 so I l 2 I I 3 I IPERIOD OF 0 I I INCUBATION 3 20 I (30 MINUTES) 2 I a. I I V I I l I I0 II I I I I I O I I I I l I I I I I ALKALIN E PHOSPHATASE CONCENTRATION(Microgroms Per Milliliter) INVENTOR- ACTIVITY INDEX R. G. SANDERS3,451,893

METHOD OF RAPIDLY DETECTING MICROORGANISMS June 24, 1969 Filed Sept. 27,1966 Sheet 2 0r 4 40 o F 8 36- 3 E 32- O. f} 28- '2 24- '5 ALKALINEPHOSPHATASE 2 CONCENTRATION. :3 k 0.039 MICROGRAMS 3 30 PER ML. C 8 E 24- o 2 6 IO l4 I8 22 26 30 TIME MINUTES FIG 6 Q 25- Z 3 8 20- E 2 0.79COUNTS s m 2 PER LITER OF AIR :0 IS- 3 w i 3 I0 Z i l 4 5 i r 'l o z a o5 IO l5 7 TIME" MINUTES ye- 7 ACTIVITY June 24, 1969 R. c. SANDERS3,451,893

I METHOD OF RAPIDLY DETECTING MICROORGANISMS Filed Sept. 27. 1966 Sheet3 of 4 Fla 4 0.038y PHOSPHATASE INDEX 4 l0 Sm 0 2 4 6 8 IO l2 TIMEMINUTES INVENTOR 2.6 sq/vases 4 0.038) PHOSPHATASEHO Sm June 24, 1969 R.G. SANDERS 3,451,893

METHOD OF RAPIDLY DETECTING MICROORGANISMS Filed Sept. 27, 1966 Sheet 4of 4 0.075 y PHOSPHATASE 2o 0.075y PHOSPHATASE 00756 PHOSPHATASETIME-MINUTES United States Patent 3,451,893 METHOD OF RAPIDLY DETECTINGMICROORGANISMS Robert G. Sanders, Minneapolis, Minn., assignor to LittonSystems, Inc., Beverly Hills, Califi, a corporation of Maryland FiledSept. 27, 1966, Ser. No. 582,275 Int. Cl. C12k 1/06, 1/10 U.S. Cl.195--103.5 9 Claims ABSTRACT OF THE DISCLOSURE This invention relates toa method of rapidly detecting microorganisms and, in particular, to arapid method for detecting microorganisms by adding a sample ofmicroorganism to an enzyme-substrate system which is poised for rapidresponse to enzymes present in the microorganism sample.

As industrial manufacturing techniques have become more complex,increasing emphasis has been placed on maintaining industrial cleanrooms free from dust and other particles, such as microorganisms.Further, continued emphasis has been placed on maintaining such rooms ashospital operating rooms and medical clinic examination rooms free frommicroorganisms such as pathogenic bacteria, fungi, rickettsia, andviruses. Much emphasis in providing relatively germ-free atmospheres forsuch rooms has been directed toward removing aerosols and microorganisms(such as pathogens) from the atmosphere in such rooms. However, anequally important aspect in the maintenance of germ-free atmospheres insuch rooms is the detection and monitoring of pathogens in theatmosphere. Of special importance is the provision of facilities forrapidly and sensitively determining the presence of pathogens in theatmosphere in such rooms. Because there has been relatively littlenecessity for rapid and sensitive detection systems in the past, prior,fairly slow laboratory techniques have been considered adequate andrelatively little effort has been expended on the problems of increasingdetection speed during the development of scientific bacteriology.

Three basic approaches and combinations thereof may be used to analyzesamples for the detection of microorganisms. For example, one canattempt to detect the organism itself, such as a vegetative cell orspore, either microscopically or by standard bacteriological plating andgrowth techniques. Secondly, one can detect some characteristicconstituent of the organisms, such as nucleic acid, protein, lipid, etc.Also, one can attempt to detect something that the organism does, suchas hydralyzing gelatin, producing carbon dioxide, etc. The lattercategory of detection, that is, detecting something that the organismdoes, is perhaps the easiest and most rapid method of detection.

Standard bacteriological methods are far too time consuming (12-24hours) for rapid detection. Identifying constituents of microorganismsis both time consuming and entirely non-specific for organisms.Detecting something the organism does is directly related to enzymeconstituents in the living cell and is'a rapid way of detecting smallchanges in bacterial population.

Chemical detection methods, such as microbiochemical assays or enzymedetection, are more specific and sometimes diagnostic. Several chemicaltechniques, such as the catalase test or the presence of indophenoloxidase, are extremely rapid (less than 1 minute). Unfortunately,however, such methods leave much to be desired because they aresensitive only to relatively high microbial concentrations, such as inthe range of 10 to 10 organisms per ml. Further, while biologicaltechniques are inherent- 1y sensitive and specific, the time involvedbetween sample collection and readout is considerably greater (in therange of 1224 hours) than that allowable for rapid detection.

Even the most recently developed chemical detection method measuringaerosolized protein suspensions, cannot detect fewer than 10 organismsper liter of air within five minutes. In particular, the backgroundprotein in the air is 10 times greater than that of one microorganismper liter of air. Therefore, between 10 and 10 microorganisms must beavailable for detection, hence, the aerosolized protein suspensiondetection method is n t sensitive enough. Morevore, because changes inthe protein concentration in air do not indicate changes in thebacterial concentration, protein concentration is not a reliable measureof bacterial concentration.

Some microbiologists have characterized bacteria as being essentiallybatteries of enzymes. Most of the biological characteristics andactivities of bacteria are derived from the presence of certain enzymes.Thus classical diagnostic bacteriology depends upon the presonce orabsence of certain enzymes (in addition to morphology and antigenicstructure) for specific identification of bacteria. It will beappreciated that microorganisms which may be detected by the presentinvention utilize enzymes in their basic metabolic processes. Forexample, exo-enzymes utilized by bacteria are excreted by bacterialcells into their environment Where the enzymes decompose certainsubstrates in the environment. These enzymes are hydrolases whichcatalyze the hydrolytic decomposition of their nutritive substrates.

Certain enzymes are vital to substantially all microorganisms andespecially to pathogens. Thus, the detection of enzymes can be anindication of the presence of microorganisms. However, only in someinstances is it possible to determine the actual amount of enzyme bychemical or physical means. Consequently, enzyme content is usuallyassayed by measuring its activity index. By this procedure, the amountof end-product (p) produced in a given time by an enzyme acting on asubstrate is an indication of the amount of the enzyme. If the enzyme isderived from microorganisms, the activity index may be correlated withthe number of microorganisms present.

It is known that enzyme reactions are extremely rapid. Thus, theturnover number of catalase is 2.5 10 mol substrate/ per mol enzyme/perminute; and of alcohol dehydrogenase is 2x10 mol substrate/molenzyme/1mm ute. However, the rate of reaction is related to the enzymeconcentration. Thus, referring now to FIG. 1 of the drawings, a curve 15illustrates the activity index (measured in terms of the production ofend product (p)) plotted against enzyme concentration. Such rapidreaction rates are achieved only along the sloping portion 16 of thecurve where the concentration of enzyme causing the reaction is in therange of 0.1 microgram/ml. to 1 mg./ ml. for unpoised alkalinephosphatase, for example, Because the enzyme concentrations which are ofinterest in the present invention are significantly below 0.1microgram/ml., and fall in the lower, flat portion 17 of the curve, theactivity index of enzyme concentrations in this flat portion 17 of thecurve are immeasurable and are not proportional to enzyme concentrationwhen standard d tection methods are used.

Procedures for measuring the enzyme activity index used in thelaboratory generally require the addition of an enzyme sample of unknownconcentration to a given substrate. The enzyme sample and the substrateare then incubated under controlled conditions for a relatively longtime to permit production of the end-product.

Because the speed of detection of microorganisms is usually an inversefunction of the number of organisms sensed, rapid detection is assistedby the use of largevolume air samplers which concentrate the particlesin a large volume of air into small amounts of liquid. By combiningimpaction, impingement and electro-static precipitation principles, onecan concentrate on a continuous basis airborne biological particles froma high volume air flow into a low volume liquid fiow. High collectionefficiencies can be obtained by such combination of methods and arecapable of collecting particles having size ranges from 0.2 to micronsin diameter, at air flow rates from 2800 to 10,000 l./min.,concentrating the particles into liquid flow rates of 2 to 10 ml./min.Thus, if the biological organisms which are to be sampled are present ata rate of only 4 organisms per liter of air, operating at 10,000 l./min.such a sampler could collect 40,000 organisms per minute and concentratethem into 10 ml. of liquid each minute so that a sample concentration of4X10 organisms per ml. would be achieved. On the other hand, prior-artcollectors such as AGI impingers, Casella samplers, etc., would requiremore than five hours to acquire sufficient organisms for so-called rapiddetection.

Research into ways to decrease the time and increase the sensitivity ofdetection using the enzymatic method of detecting biological organismsindicates that a rapid method of detecting such organisms is to poise anenzyme-substrate system by mixing with a substrate that amount of enzymerequired to maintain an equilibrium between the enzyme, the substrateand an enzyme-substrate complex produced by the action of the enzyme onthe substrate. At this critical equilibrium point in the reaction, theaddition of a trace amount of free enzyme to the enzyme-substrate systemwill cause a sudden shift in the equilibrium and the formation of asubstantial detectable amount of end-product. By proper selection of thepoising amount of enzyme, one can determine the smallest amount of freeenzyme effecting the formation of the greatest amount of end-product inthe shortest period of time. With such poised enzyme-substrate system,minimum concentrations (10 organisms/ml.) of microorganisms can bedetected in about five minutes.

An object of the present invention is to provide a new and improvedmethod of rapidly and sensitively detecting microorganisms by detectingenzymes which are vital to such microorganisms.

Another object of the present invention is to provide a method ofpoising an enzyme-substrate system in an equilibrium condition andadding thereto microorganisms to be detected, wherein minimumconcentrations of enzymes of such microorganisms cause the production ofa detectable amount of end-product.

A further object of the present invention is to provide a method ofmaking a poised enzyme-substrate system which includes selected amountsof alkaline phosphatase and disodium-phenyl-phosphate for rapidlydetecting minute amounts of the enzyme alkaline phosphatase which may bepresent in microoragnisms to be detected.

With these and other objects in view, the present invention contemplatesa significant change in the standard enzyme activity detectionprocedures in which an enzyme to be detected is added directly to asubstrate. In particular, the present invention contemplates using apoised enzyme-substrate system. The enzyme-substrate system is formed bycombining with a given amount of a substrate, that amount of enzymerequired to maintain an equilibrium between the enzyme, the substrateand the enzyme-substrate complex. Rather than adding free enzyme to bedetected to only the substrate, the free enzyme is added to the poisedenzyme-substrate system. When the enzyme-substrate is poised, theaddition of only a trace amount of free enzyme causes a sudden shift inthe equilibrium and the formulation of a significant, measurable amountof end product.

A complete understanding of the present invention may be had byreferring to the following detailed description and the accompanyingdrawings illustrating various aspects thereof, wherein FIG. 1 is a graphof activity index plotted against enzyme concentration illustrating thevery low, relatively constant rate of formation of an end product at lowenzyme concentrations;

FIG. 2 is a graph of activity index plotted against enzyme concentrationillustrating a plateau formed when a poising amount of enzyme is used;

FIG. 3 is a graph illustrating activity index plotted against reactiontime when a poising amount of enzyme is used;

FIG. 4 is a graph of activity index plotted against reaction timemeasuring alkaline phosphatase activity of the microorganism Serratz'amarcescens (Sm) with and without poising;

FIG. 5 is a graph of activity index plotted against reaction timemasuring alkaline phosphatase activity of the microorganisms Bacillussubtilis var niger (Bg) with and without poising;

FIG. 6 is a bar graph showing the change in activity level of anenzyme-substrate system which is poised for maximum response toorganisms in the shortest possible time.

The method of the present invention uses an enzymesubstrate system whichis poised for rapid response to a minimum amount of enzymes present inmicroorganisms which are to be detected. Because certain enzymes, suchas alkaline phosphatase, for example, are common to all microorganisms,except for Rickettsia and viruses, such enzymes may be used as aconstituent of the enzyme-substrate system of the present invention. Inparticular, the enzyme alkaline phosphatase has been shown to be anexcellent enzyme when used as an alkaline phosphatase suspension inammonium sulfate, such suspension being hereinafter referred to as anenzyme suspension. The enzyme alkaline phosphatase may be extracted fromType III E. coli available from the Sigma Chemical Co., for example.

A substrate illustrating the principles of the present invention isdisodium-phenyl-phosphate which is available, for example, from theApplied Research Institute in compressed tablet form under the tradename of Stabilized Phos-Phax. A suitable preparation ofdisodiumphenyl-phosphate may be made by dissolving one such tablet in 50ml. water to yield a concentration of 0.50 mg./ml. ofdisodium-phenyl-phosphate in 0.0745 M. borate buffer pH 9.6. Suchsolution of a substrate will hereinafter be referred to as the substratesolution.

The enzyme suspension and the substrate solution are mixed usingselected concentrations of each to provide the poised enzyme-substratesystem. The selected concentration of enzyme suspension is determined byproviding a number of containers, each holding the same given quantityof substrate solution at a given concentration (i.e. 2.5 mg./ml.) whichis in excess of that required to react with the enzyme suspension to beadded and at the optimum pH (i.e. 9.5) for reaction. To such containersof substrate solution are added equal quanti ties of enzyme suspensionhaving varying enzyme concentrations. The mixtures are incubated atconstant temperature, such as 37 C., for a given time period, such as 30minutes. The activity index (in terms of the amount of end productproduced) is determined at the end of the given time period.

Reference to FIG. 2 will illustrate the results of this procedure. InFIG. 2, there is shown a curve 20 representing the activity index (interms of end product 1) produced during the given incubation period) forvarious concentrations of alkaline phophatase. As shown in FIG. 2, thereis a distinct plateau 22 in the curve 20. In particular, the activityindex is relatively constant for alkaline phosphatase concentrationsbetween 3.75 and 5.75 X10 micrograms per ml.

The significance of the plateau 22 may be appreciated by considering thereaction between the enzyme suspension and the substrate solution.

E=enzyme suspension S=substrate solution ES=enzyme-substrate complexconcentratlon P=end product then, where the activity index plateau 22occurs, the rate of formation of end product (P) is a minimum and therate of formation of the enzyme-substrate complex (ES) is maximum. Thisindicates that the enzyme-substrate system is poised when enzymeconcentrations are between 3.75 and 5.75 X 10 micrograms per ml.

Referring to FIG. 3, a curve 30 illustrates the critical nature of theenzyme concentration-time relationship favorable to the formation of theenzyme-substrate complex (ES). When the enzymesub-strate system having aconcentration of 0.039 microgram per ml. of alkaline phosphatase wasused, the activity index remained sub stantially unchanged for 10minutes along a portion 32 of the curve 30 between 4 and 14 minutes. Theactivity index then doubled in the next 10 minutes. The curve 30illustrate optimum conditions for the formation of the enzyme-substratecomplex (ES) during the 4 to 14 mmute period in which there was nosubstantial change in the production of end product (P).

When the enzyme-substrate system includes the enzyme alkalinephosphatase and the substrate disodiumphenyl-phosphate, the addition oftrace amounts of microorganisms causes the enzyme to hydrolyze thesubstrate and produce measurable amounts of the end product (P) phenol.The Sharer phosphatase procedure may be used to determine the amount ofphenol produced. In this procedure, 2,6 dibromo-quinone-chloroimide(BQC) is added to a container holding the microorganisms and the poisedsystem for reaction with the phenol to produce indophenol, which is bluein color. The intensity of the color, which is proportional to theamount of phenol produced, is quantitatively determined optically byreading in a colorimeter at a Wave length of 650 millimicrons.

The standard Sharer procedure requires five minutes at room temperatureto develop the blue indophenol color after the addition of the BQC. Anaspect of the present invention is the modification of the Sharer methodso that the normal time may be substantially reduced without adverseeffects on the color formation. The modified Sharer procedure includesplacing the container in a boiling water bath. This method causesdevelopment of maximum blue color within 15 seconds, rather than 5minutes. It has been found that this rapid color development is achievedwhen the water bath temperature is between 95 and 100 C.

The Sharer procedure was used to determine whether or not the endproduct phenol was produced during the 4 to 14 minute portion 32 of thecurve 30 of FIG. 3. The color formed in the 4-minute reaction tube wasgreen, rather than blue, indicating the formation of a compounddifferent from phenol. Spectrophotometric analysis showed that thisgreen colored compound had a distinctly different absorption spectrum.Thus, it is clear that at the level portion 32 of the curve 30 of FIG.3, there is momentary isolation of the enzyme-substrate complex (ES).

The significance of the plateau in the activity index vs. enzymeconcentration curves 15 and 20 of FIGS. 1 and 2 is shown in FIG. 4.Referring to FIG. 4, the activity index (in terms of production of endproduct (P)) is shown plotted against time for various concentrations ofthe microbial agent Serratia marcescens (Sm). The enzyme-substratesystem used in these tests included an enzyme suspension of alkalinephosphatase having a concentration of 0.038 microgram of enzyme per ml.and the substrate solution of disodiumphenyl-phosphate referred toabove.

To illustrate the nature of the reaction resulting from only the Smsamples, curve 40 shows the activity index of the Sm samples alone.Curve 40 indicates the activity index to be below 5, hence it is withinthe range of experimental error and is not reliable.

Also, to illustrate the sensitivity of the poised enzymesubstrate systemto the Sm samples, reference is made to curves 42 and 44. Curve 42 showsthe activity index of the poised enzyme-substrate system without the Smsamples, whereas curve 44 shows the significantly increased activityindex when 10 Sm microbial agents were added to the poisedenzymephosphatase system. These curves 42 and 44 indicate a significantdifference in activity index after 5 and 10 minutes incubation time.This difference is 4 after 5 minutes and 9 after 10 minutes.

In FIG. 5, there are shown a series of curves showing the effect ofadding lyophilized cultures of Bacillus subtilis var niger (Bg) ofvarious concentrations to poised" and unpoised enzyme systems.

The enzyme-substrate system in this example included the enzyme alkalinephosphatase having a concentration of 0.0756 mg. per ml. Curve 50illustrates the very low activity index which results from the additionof 10 microorganisms of Bg to unpoised alkaline phosphatase. Curve 52shows the activity index of the poised enzyme-substrate system. Theeffect of adding 750 Bg organisms to the poised enzyme-substrate systemis shown in curve 54, whereas the effect of adding 7500 Bg organisms isshown in curve 56. Curves 54 and 56 clearly show that measurable amountsof end product (P) are produced by the poised enzyme system when only750 Bg organisms are being detected as compared to immeasurable amountsof end product (P) which are produced by 1000 Bg organisms addedaccording to prior art techniques to an unpoised substrate (curve 50).

The enzyme-substrate system of the present invention may be formed usingthe substrate para-nitro-phenylphosphate. This substrate is a compoundwhich, upon hydrolysis of the phosphate group by phosphatase, yields theyellow salt of para-nitro-phenol having a characteristic absorptionmaximum of 400 millimicrons. To prepare a substrate solution using thissubstrate, one hundred mg. of Sigma 104 para-nitro-phenyl-phosphatesubstrate is dissolved in 25 ml. of water. Equal parts of this solutionand Sigma Alkaline Buffer solution are mixed to produce a substratesolution which may be added to the alkaline phosphatase enzymesuspension to form the enzyme-substrate system.

The poised enzyme-substrate system is formed by adding one (1) ml. ofthe para-nitro-phenyl-phosphate substrate solution to one (1) ml. of thealkaline phosphatase enzyme suspension having an enzyme concentration ofbetween 0.15 and 0.25 microgram per ml. Because the rate of hydrolysiswas slow, the para-nitro-phenylphosphate substrate is more diflicult topoise and is less useful than the disodium-phenyl-phosphate.

The step of the present invention relating to collecting a sample ofmicroorganisms can be performed by apparatus such as that disclosed inthe I. L. Milton Patent 2,336,625 when the microorganisms are airborne.This apparatus includes an air intake provided with corona dischargepoints which ionize the microorganisms. The air and microorganismsimpact against a film of liquid which flows across a disc which isrotated at a rate of 500 rpm, for example. An electric field providedadjacent the disc urges the ionized microorganisms toward the disc andinto the flowing liquid.

If microorganisms are present in the air in quantities of 4 organismsper liter of air and if such apparatus takes in 10,000 liters of air perminute, it can collect 40,000 organisms per minute. The liquid flow ratecan be adjusted to 10 ml. per minute so that a microorganism sample of 410 organisms per minute is collected. This concentration of organisms iswell within the rapid detection capability of the present method.

While the type of apparatus disclosed in the Milton patent is suitablefor the collection of inorganic matter, such as dust and other airborneparticles, certain critical modifications must be made to precludeundesired effects on the viable microorganisms which may be detected bythe poised enzyme-substrate system of the present invention.

In particular, the liquid used to collect the microorganisms must notprovide a variable level of activity index, but should provide aconstant background against which the activity index of themicroorganisms may be measured. Moreover, the liquid must adequately wetthe collection disc and must be compatible with both the microorganismsto be detected and the phosphatase used in the poised system.Investigation of liquids which meet these requirements indicates that aliquid suitable for collecting microorganisms to be detected is amixture of water and between 0.1 and 1.0 percent of non-ionicpolyoxyethylene-lauryl-ether, such as that sold under the trade nameBrij-35 by the Atlas Chemical Co.

In the use of this liquid, the collection disc may be manually wet withthe water mixture, whereafter a liquid input of about 11 ml. per minuteis suitable for maintaining the disc wet under an air flow rate of10,000 l./minute and with standard relative humidity conditions. Thismixture does not inhibit the growth of microorganisms, such as Sm andBg, for example, and does not adversely effect the phosphatase activityof the poised system.

Reference is made to FIG. 6 wherein a bar graph illustrates the changein activity index at zero, five, ten and fifteen minute incubationperiods of 0.79 Sm organisms per liter of air added to a poised enzymesubstrate system. The system included the enzyme alkaline phosphatasehaving a concentration of 0.0680 mg./ml., the incubation was performedat 37 C., and the system was poised to detect the smallest quantity ofphosphatase by giving a maximum response in the shortest possible time.The activity units shown in the graph represent the difference betweenthe activity of the poised system plus the microorganism sample and theactivity of the poised system plus the background activity level of thecollection liquid, which was the water mixture. FIG. 6 shows that thesystem was poised for maximum response to as few as 0.79 Sm organism perliter of air. The response was maximum in five minutes as shown by deltaA of approximately 24 after a -minute incubation period.

Other experiments conducted in this manner indicate that as few as 0.5organism per liter of air may be detected within 7 minutes from the timethe organisms are introduced into the sampler.

It is to be understood that the above-described arrangements are simplyillustrative of the application of the principles of this invention.Numerous other arrangements may be readily devised by those skilled inthe art which will embody the principles of the invention and fallwithin the spirit and scope thereof.

What is claimed is:

1. The method of rapidly detecting microorganisms by response to enzymesof such microorganisms, which comprises;

collecting a sample of microorganisms;

providing a poised enzyme-substrate system which comprises a givenenzyme common to the microorganism of said sample and a selectedsubstrate, said substrate and enzyme being mixed in predeterminedconcentrations to provide an equilibrium condition with respect to theproduction of an enzyme-substrate complex said condition beingcharacterized by the production of minimum amounts of an end product;

adding at least a portion of said sample to said poised enzyme-substratesystem to rapidly produce measurable amounts of said end product; and

measuring the amount of end product produced to indicate the presence ofsaid microorganisms.

2. The method of claim 1, wherein:

said given enzyme is a phosphate esterase.

3. The method of claim 1, wherein:

said given enzyme is alkaline phosphatase.

4. The method of claim 3, wherein:

said alkaline phosphatase is an alkaline phosphatase suspension inammonium sulfate having a phosphatase concentration of between 0.0375and 0.0575 mg./m1. 5. The method of claim 1, wherein: said selectedsubstrate comprises a given volume of disodium-phenyl-phosphate having aconcentration of about 50 mg./ml. in 0.0745 mol borate buffer and a pHof about 9.6, and wherein said given enzyme comprises said given volumeof alkaline phosphatase suspension in ammonium sulfate and having aphosphatase concentration of between 0.0375 and 0.0575 mg./ml. 6. Themethod of claim 5, wherein said step of measuring the amount of endproduct produced is performed by:

adding a selected amount of 2,6-dibromo-quinonechloroimide to acontainer having therein said microorganism sample and enzyme-substratesystem, and

placing said container in a boiling water bath to rapidly develop blueindophenyl to indicate the presence of said microorganisms.

'7. The method of claim 1, wherein:

said selected substrate is selected from the group consisting ofpara-nitro-phenyl-phosphate and disodiumphenyl-phosphate.

8. The method of poising a mixture of an enzyme and a substrate, whichcomprises the steps of:

providing a plurality of containers each holding the same quantity of agiven substrate at a concentration which is in excess of that requiredto react with said enzyme;

providing a series of mixtures by adding to said containers equalquantities of said enzyme having different concentrations;

incubating said mixtures at constant temperature for a given period oftime;

at the end of said period of time determining the amount of an endproduct produced by the action of said enzyme on said substrate;

determinig the range of those enzyme concentrations which producesubstantially the same amounts of end product in said period of time;and

mixing said substrate with said enzyme having a concentration withinsaid range of concentrations to produce said poised enzyme-substratesystem.

9. The method of claim 8, wherein:

said given substrate is selected from the group consisting ofpara-nitro-phenyl-phosphate and disodiumphenyl-phosphate, and

said enzyme is alkaline phosphatase.

(References on following page) 9 10 References Cited Dixon et a1.:Enzymes, 2nd edition pp. 101411 UNITED STATES PATENTS 2359952 9/1944Schmr ALVIN E. TANENHOLTZ, Primary Examiner. OTHER REFERENCES 5 US Cl XR Bayer et al.: The Enzymes, v01. 1, 2nd edition pp. 19,

Qt 'm UNI'IEI) S'IA'HCS r/mewr owuzi-z (IICR'IH ICA'EE (W CORRECTIONInventork.) R G, SANDERS It is certified Lhat error appears; in the.above-identified patent and that said Letters Patent are herebycorrected as shown below:

In the specification, Column 4, Line 6, after the word substrate, insert--system--.

In the specification, Column 5, Line 1 after the word solution, insert--If the reaction is represented as follows:--.

SIGNED AND SEALED Nov 4 1959 (SEAL) Attest:

Edward M. Fletcher, Jr. WILLIAM E. summons, JR.

Commissioner of Patents Attesting Officer

